Biological systems universally employ cascades of binding/catalytic events to transmit information regarding environmental and physiological states. This communication network requires that protein function be altered by allosteric mechanisms. The dearth of atomic level insight into such intramolecular signal transduction motivates our continued studies of the "switching" pathway in the well-studied lactose repressor protein (LacI). In LacI function, ligand binding information must flow through the protein structure between the widely separated inducer and DNA binding sites. Our ultimate goal is an atomic-level view and sequential timeline of the conformational changes that result in release of operator DNA and consequent transcription of downstream genes. We recently used targeted molecular dynamics simulation (TMD) to predict an atomic-level allosteric pathway that is concordant with the large library of experimental data on LacI. Coupled with available phenotypic information, this model provides a unique framework for designing novel experiments to examine and redesign the conformational behavior of LacI and to evaluate TMD as a general tool for deciphering allosteric pathways. Experiments are proposed to examine specific features of LacI: (1) the potential for a single (or only a few) amino acid(s) to trigger allosteric response to inducer; (2) the role of the core N-subdomain interface in adopting the alternate conformations; (3) the regulation of structural shifts by amino acids in the 3-stranded core pivot that links the N- and C-subdomains of the LacI core; and (4) the influence of symmetry on the allosteric response. Site-specific mutagenesis will be used in concert with detailed biophysical analysis (and, where possible, structural data) to assess the role of specific amino acids in the allosteric pathway and the influence of asymmetric amino acid changes within a dimer. LacI provides a unique system with the requisite background of information and tools to test rigorously the predictions of TMD calculations. With established reliability, TMD can be used to more fully exploit the increasing number of end-point structures available for allosteric proteins. Finally, information on allosteric changes in LacI can be integrated with the recent successful design of novel ligand binding activities based on the protein fold common to the LacI core and other protein families. Comprehensive understanding of LacI behavior and function may allow extension of design principles to generate even more complex allosteric systems. [unreadable] [unreadable]